Construction of new xylose utilizing saccharomyces cerevisiae strain

ABSTRACT

The present invention relates to a novel  Saccharomyces cerevisiae  strain utilizing xylose for fermenting ethanol expressing xylose isomerase (XI), overexpressing xylulokinase (XK), overexpressing the pentose phosphate pathway (PPP), and non-expressing aldose reductase (AR) and being adapted to growth in mineral defined medium with xylose as sole carbon source.

The present invention relates to a novel Saccharomyces cerevisiae strainproducing ethanol from xylose containing medium.

BACKGROUND OF THE INVENTION

Production of ethanol for use as e.g., fuel or fuel additive fromcarbohydrate feedstocks, such as hydrolysates of plants can be made byethanolic fermentation using yeasts, such as Saccharomyces cerevisiae.As such feedstocks may comprise pentoses, such as xylose there is ademand for Saccharomyces cerevisiae strains that can convert not onlyhexoses but also pentoses such xylose.

Lignocellulose is the main component of forest product residues andagricultural waste. Lignocellulosic raw materials are mainly composed ofcellulose, hemicellulose, and lignin. The cellulose fraction is made upof glucose polymers, whereas the hemicellulose fraction is made up of amixture of glucose, galactose, mannose, xylose, and arabinose polymers.The lignin fraction is a polymer of phenolic compounds.

The cellulose and hemicellulose fractions can be hydrolyzed to monomericsugars, which can be fermented to ethanol. Ethanol can serve as anenvironmentally friendly liquid fuel for transportation, since carbondioxide released in the fermentation and combustion processes will betaken up by growing plants in forests and fields.

The price for lignocellulose-derived ethanol has been estimated by vonSivers et al. (“Cost analysis of ethanol production from willow usingrecombinant Escherichia coli, Biotechnol. Prog. 10:555-560, 1994). Thecalculations are based on the fermentation of all hexose sugars(glucose, galactose, and mannose) to ethanol. It was estimated that thefermentation of pentose sugars (xylose and arabinose) to ethanol willreduce the price of ethanol by approximately 25%.

Xylose is found in hardwood hemicellulose, whereas arabinose is acomponent in hemicellulose in certain agricultural crops, such as corn.In order to make the price of ethanol more competitive, the price mustbe reduced.

The release of monomeric sugars from lignocellulosic raw materials alsoreleases by-products, such as weak acids, furans, and phenoliccompounds, which are inhibitory to the fermentation process. Numerousstudies have shown that the commonly used Baker's yeast, Saccharomycescerevisiae, is the only ethanol producing microorganism that is capableof efficiently fermenting non-detoxified lignocellulose hydrolysates(Olsson and Hahn-Hägerdal, “Fermentation of lignocellulosic hydrolysatesfor ethanol production”, Enzyme Mjcrobial Technol. 18:312-331 (1996).Particularly efficient fermenting strains of S. cerevisiae been isolatedfrom the fermentation plant at a pulp and paper mill (Linden et al.,“Isolation and characterization of acetic acid-tolerantgalactose-fermenting strains of Saccharomyces cerevisiae from a spentsulfite liquor fermentation plant”, Appl. Envjron. Mjcrobjol.58:1661-1669, 1992).

S. cerevisiae ferments the hexose sugars glucose, galactose and mannose,but is unable to ferment the pentose sugars xylose and arabinose due tothe lack of one or more enzymatic steps. S. cerevisiae can fermentxylulose, an isomerisation product of xylose, to ethanol (Wang et al.,“Fermentation of a pentose by yeasts”, Biochem. Biophys. Res. Commun.94:248-254, 1980; Chiang et aJ., “D-Xylulose fermentation to ethanol bySaccharomyces cerevisiae”, Appl. Environ. Microbiol. 42:284-289, 1981;Senac and Hahn-Hägerdal, “Intermediary metabolite concentrations inxylulose- and glucose-fermenting Saccharomyces cerevisiae cells”, Appl.Environ. Microbiol. 56:120-126, 1990).

In eukaryotic cells, the initial metabolism of xylose is catalyzed by axylose reductase (XR), which reduces xylose to xylitol, and a xylitoldehydrogenase (XDH), which oxidizes xylitol to xylulose. Xylulose isphosphorylated to xylulose 5-phosphate by a xylulose kinase (XK) andfurther metabolized through the pentose phosphate pathway and glycolysisto ethanol. S. cerevisiae has been genetically engineered to metabolizeand ferment xylose via this pathway. The genes for XR and XDH from thexylose fermenting yeast Pichia stipitis have been expressed in S.cerevisiae (European Patent to C. Hollenberg. 1991; Hallborn et al.,“Recombinant yeasts containing the DNA sequences coding for xylosereductase and xylitol dehydrogenase enzymes”, WO91/15588; Kbtter andCiriacy, “Xylose fermentation by Saccharomyces cerevisiae”, Appl.Microbiol. Biotechnol. 38:776-783, 1993). The transformants metabolizexylose but do not ferment the pentose sugar to ethanol.

When the gene for the enzyme transaldolase (TAL) is overexpressed inxylose-metabolizing transformants, the new recombinant strains growbetter on xylose but still do not produce any ethanol from xylose(Walfridsson et al., “Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TAL1 genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase”, Appl.Environ. Microbiol. 61:4184-4190, 1995). In these strains, the majormetabolic by-product, in addition to cell mass, is xylitol formed fromxylose through the action of the enzyme XR. When the expression of XDHis ten times higher than the expression of XR, xylitol formation isreduced to zero (Walfridsson et al., “Expression of different levels ofenzymes from Pichia stipitis XYL1 and XYL2 genes in s and its effect onproduct formation during xylose utilization”. Appl. Microbiol.Biotechnol. 48:218-224, 1997). However, xylose is still poorly fermentedto ethanol.

The gene for xylulose kinase (XK) from S. cerevisiae has been cloned andoverexpressed in XR-XDH-expressing transformants of S. cerevisiae (Dengand Ho, ‘Xylulokinase activity in various yeasts including Saccharomycescerevisiae containing the cloned xylulokinase gene”, Appl. Biochem.Biotechnol. 24125.193-199, 1990. Ho and Tsao, “Recombinant yeasts foreffective fermentation of glucose, and xylose”, WO95/13362, 1995;Moniruzzaman et al., “Fermentation of corn fibre sugars by an engineeredxylose utilizing Saccharomyces strain”, World J. Microbiol. Biotechnol.13:341-346, 1997). These strains have been shown to produce netquantities of ethanol in fermentations of mixtures of xylose andglucose. Using the well established ribosomal integration protocol, thegene have been chromosomally integrated to generate strains that can beused in complex media without selection pressure (Ho and Chen, “Stablerecombinant yeasts for fermenting xylose to ethanol”, WO97/42307. Toonet al., “Enhanced cofermentation of glucose and xylose by recombinantSaccharomyces yeast strains in batch and continuous operating modes”,Appl. Biochem. Biotechnol. 63/65:243-255, 1997).

In prokaryotic cells, xylose is isomerized to xylulose by a xyloseisomerase (XI). Xylulose is further metabolized in the same manner as inthe eukaryotic cells. XI from the thermophilic bacterium Thermusthermophilus was expressed in S. cerevisiae, and the recombinant strainfermented xylose to ethanol (Walfridsson et aJ., “Ethanolic fermentationof xylose with Saccharomyces cerevisiae harboring the Thermusthermophilus xylA gene which expresses an active xylose (glucose)isomerase”, Appl. Environ. Microbiol. 62:4648-4651, 1996). The low levelof ethanol produced was assumed to be due to the fact that thetemperature optimum of the enzyme is 85° C., whereas the optimumtemperature for yeast fermentation is 30° C.

Saccharomyces cerevisiae as such can thus not ferment xylose, but has tobe modified. Thus one way is to overexpress the genes coding for xylosereductase (XR), xylitol dehydrogenase (XDH) and xylulokinase (XK),whereby and isomerisation product of xylose, viz. xylulose, is obtained.

Another way is to overexpress xylose isomerase (XI), whereby xylose isdirectly converted to xylulose. (Träff et al, Appl Environ Microbiol2001:67(12):5668-74; Lönn et al, Enz Microbiol Tech, 2003:32:567-573).Hereby a recombinant S. cerevisiae strain comprising mutated xylA fromThermus thermophilus is construed.

Kuyper et al, FEMS Yeast Res 2003:1574:1-10 discloses high-levelfunctional expression of fungal xylose isomerase derived from Piromycesxylose isomerase gene. The strain construed was not shown to growanaerobically or aerobically on a glucose-xylose medium but show a smallxylose uptake. The strain grew on sole xylose with a growth rate of0.005.

However, this strain utilizes a combined glucose-xylose medium, andseems not to be adapted to a mere xylose medium.

There is thus a demand for a xylose fermenting strain expressing xyloseisomerase and having an improved growth rate and improved ethanol yield.

SUMMARY OF THE INVENTION

In accordance with the present invention a new Saccharomyces cerevisiaestrain has been construed solving this problem. The strain comprises axylose isomerase (XI) expressing gene xylA disclosed in Lönn et al(supra) but also present in plasmid pBXI, an overexpression ofxylulokinase (XK), an overexpression of the pentose phosphate pathway,having a deleted GRE3 gene (Träff et al, supra) and being adapted togrowth in mineral defined medium with xylose as the sole carbon source.

The XI used originated from a plasmid PBXI, and is thus a wild-type XI.

This Saccharomyces cerevisiae strain denoted TMB 3050, has beendeposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen onthe 14^(th) of August 2003, under deposition number DSM 15834.

DETAILED DESCRIPTION OF THE INVENTION

The present invention thus claims a Saccharomyces cerevisiae strainexpressing xylose isomerase (XI), overexpressing xylulokinase (XK),overexpressing the pentose phosphate pathway, non-expressing aldosereductase (AR) and being adapted to growth in mineral defined mediumwith xylose as sole carbon source.

In particular the strain expresses xylose isomerase derived from xylAgene. Overexpression of xylulokinase is obtained by adding a plasmidexpressing XKS1 (Lönn et al. 2003) coding for xylulokinase.Overexpression of the pentose phosphate pathway is obtained by addingextra copies of the genes TAL1, TKL1, RPE1, RKI1 (Johansson &Hahn-Hägerdal 2002). Non-expression of aldose reductase (AR) is obtainedbe deleting the gene GRE3, to reduce formation of xylitol.

The construction of the strain will be described more in detail in thefollowing.

Methods

Standard molecular biology techniques were used. (Sambrook et al. 1989)Lithium acetate method was used for yeast transformation (Gietz &Schiestl 1995). Yeast chromosomal DNA was extracted with Easy-DNA Kit(Invitrogen). Colony PCR was performed using the Lyse-N-Go reagent(Pierce, Rockford, Ill., USA). PCR was performed with the followingprogram: 95° C. for 5 min, 45 cycles of 95° C. for 30 s, 45° C. for 30 sand 72° C. for 1 min 20 s, 72° C. for 7 min. In colony PCR and PCR onchromosomal yeast DNA, Taq-polymerase (Fermentas) was used. In other PCRreactions, PWO polymerase (Roche) was used.

Media and Cultivation Conditions

Yeast was grown on YPD medium (20 g/l peptone, 10 g/l yeast extract and20 g/l glucose), SC medium (6,7 g/l Difco Yeast Nitrogen Base, 20 g/lglucose or 20 g/l galactose) or defined mineral medium (Verduyn et al.1990). The amount or sugar used in mineral medium was 20 g/l glucose or50 g/l xylose. 2.042 g phthalate and 0.301 g NaOH was added to mineralmedium, and pH was set to 5.5 before sterilization. Amino acids wereadded to defined mineral medium when necessary. The amino acidconcentrations used were: 20 μg/ml histidine, 20 μg/ml tryptophan, 240μg/ml leucine and 20 μg/ml uracil. The cultures were grown in baffledshake flasks with 130 rpm shaking.

Plate cultures were grown on YPD-agar plates (20 g/l peptone, 10 g/lyeast extract and 20 g/l glucose, 20 g/l agar) or YNB-plates (6,7 g/lDifco Yeast Nitrogen Base, 30 g/l agar and 20 g/l glucose or 50 g/lxylose). Zeocin (Invitrogen, Groningen, The Netherlands) was added toYPD plates at 50 mg/l.

Construction of TMB 3044

The Saccharomyces cerevisiae strain YUSM1009a (Träff et al. 2001) wastransformed with plasmid YIpXK (Lönn et al. 2003) linearized with NdeI.Transformants were selected on YNB-plates containing uracil, leucin andtryptophan but not histidine. Chromosomal integration of the plasmid wasconfirmed by colony PCR and PCR on chromosomal DNA with primers BJ0697and BJ5756. The overexpression of the gene coding for xylulokinase (XK)was confirmed with enzyme assay.

The resulting strain was transformed with plasmid pB3PGK TAL1 (johansson& Hahn-Hägerdal 2002) linearized with BglII. Transformants were selectedon YPD plates containing zeocin. Chromosomal integration of the plasmidwas confirmed by colony PCR and PCR on chromosomal DNA with primersBJ5756 and 3TAL1clon.

To remove the zeocin marker, the resulting strain was transformed withplasmid pCRE3 (Johansson & Hahn-Hägerdal 2002). The transformants wereselected on YNB plates containing leucin and tryptophan, but not uracil.The resulting transformant was grown in 500 ml shake flask in 100 mlSC-medium containing galactose for about 24 h. To remove the plasmid, 1ml aliquot of the culture was inoculated to 100 ml YPD medium in 500 mlshake flask and grown for 24 h. An aliquot of the culture was plated ona YPD plate. Zeocin-sensitive colonies were selected by replica platingon a YPD plate containing zeocin. One zeocin sensitive clone waspurified by repeated plating on YPD plates.

The resulting clone was transformed with pB3PGK RKI1 (Johansson &Hahn-Hägerdal 2002) linearized with BcuI (Fermentas). Chromosomalintegration of the plasmid was confirmed by colony PCR with primersBJ5756 and 3RKI1clon. The zeocin marker was removed same way as before.

The resulting clone was transformed with pB3PGK TKL1 (Johansson &Hahn-Hägerdal 2002) linearized with BshTI (Fermentas). Chromosomalintegration of the plasmid was confirmed by colony PCR with primersBJ5756 and 3TKL1clon. The zeocin marker was removed same way as before.Overexpression of the pentose phosphate pathway is thereby obtained.

The resulting clone was transformed with pB3PGK RPE1 (Johansson &Hahn-Hägerdal 2002) partially digested with XcmI (New England Biolabs).Chromosomal integration of the plasmid was confirmed by colony PCR withprimers BJ5756 and 3RPE1clon. The zeocin marker was removed same way asbefore.

Tryptophan auxotrophy in the resulting strain was cured by transformingwith product from a PCR with primers TRP5 and TRP3 and the plasmidYEplac112 as a template. The transformants were selected on a YNB platelacking tryptophan.

In the resulting strain, leucin auxotrophy was cured by transformingwith the plasmid YEplac181 linearized with ScaI. The transformants wereselected on a YNB plate lacking leucin. The resulting strain was namedTMB 3044.

Plasmid Construction

A cassette of HXT7 truncated promoter and PGK terminator was digestedfrom plasmid pHM96 (Hauf et al. 2000) with Sad and HindIII. Theresulting fragment was cloned in YEplac195 linearized with SacI andHindIII. The resulting plasmid was named YEplacHXT.

The xylose isomerase gene xylA of Thermus thermophilus was amplified byPCR using primers prBCL and terPST and plasmid pBXI (Walfridsson et al.1996) as a template. The product was digested with BclI and PstI andcloned in plasmid YEplacHXT linearized with BamHI and PstI. Theresulting plasmid was named YEplacHXT-XI to express xylose isomerasewhen inserted.

The resulting plasmid was named YEplacHXT-XI, having T. thermophilusxylA gene downstream of the HXT7-truncated promoter for highest possibleexpression of T. thermophilus XI.

Construction of TMB3050

Plasmid YEplacHXT-XI was transformed to TMB 3044. Transformants wereselected on YNB plates lacking uracil. One of the transformants waspurified by repeated plating on YNB plates. The purified transformantwas grown in mineral medium containing glucose and an aliquot of theculture was plated on an YNB plate containing 50 g/l xylose as a solecarbon source. After two months of incubation at 30° C., about 20 of the˜1000 colonies on the plate appeared larger than others. One of thesecolonies was purified by repeated plating on a YNB plate containing 50g/l xylose. After purification, the strain was grown in mineral medium(50 g/l xylose, no phthalate, no NaOH) for 4 weeks. An aliquot of theculture was reinoculated to fresh medium and the culture was incubatedfor two weeks. When an aliquot of this culture was re-inoculated, theculture reached in three days stationary phase at optical density (620nm) of 7.7. An aliquot of this culture was purified by repeated plating.

This culture was named TMB 3050. When grown on mineral medium containing50 g/l xylose, buffered with phthalate and NaOH, the strain grows withmaximal growth rate of 0.12-0.14 and reaches optical density of >15 inabout 3 days (FIG. 1).

FIG. 2 shows the gene construct of the present strain

The present strain was compared with the strains according to Kuyper etal (literature comparison) and the strain of Lönn et al (supra) as toaerobic growth, and anaerobic growth. TABLE 1 TMB3050 Lönn et al Kuyperet al AEROBIC Growth rate 0.12-0.14 n.a. 0.005 Xylose uptake rate 0.1466n.a. 0.0495 (g xylose/g cells/h) XI activity 0.23 0.012 0.4-1.1 (U/mgcell extract) (50° C.) (30° C.) (30° C.) ANAEROBIC Temperature at 30° C.40° C. 30° C. fermentation Type of fermentation High cell density Highcell density Chemostat Sugar composition 40 g/l xylose 50 g/l xylose 20g/l glucose + in media 10 g/l xylose Xylose uptake rate 0.0034 0.00430.109 (g xylose/g cells/h) Ethanol yield/ 0.0439 n.a. n.a. total xyloseEthanol yield/ 0.389 0.43 n.a. consumed xylose (wt XI) (mutant 1021)Xylitol yield/ 0.365 0 n.a. consumed xylosen.a. = not available information

As evident from Table 1 above the growth rate of the present strain is26 times higher than the growth rate of Kuyper et al, and producesethanol at 30° C., which the Kuyper et al strain does not. TABLE 2Specific xylose uptake rates (q_(xylose)), ethanol and xylitol yieldcoefficients (Y_(ethanol) and Y_(xylitol)) and specific ethanolproductivities (q_(ethanol)) from aerobic and anaerobic batchcultivations of S. cerevisiae strains TMB 3050 and TMB 3045 (=TMB 3044 +XI). Aerobic Anaerobic Strain q_(xylose) ^(b) q_(xylose) ^(b)Y_(ethanol) ^(c) Y_(xylitol) ^(d) q_(ethanol) ^(e) TMB 3050 0.16 ± 0.0170.0049 ± 0.0013 0.029 ± 0.013 0.031 ± 0.009 0.0012 ± 0.0001 TMB 3044 +XI 0 0 0 0 0^(a)h⁻¹^(b)g xylose g cells⁻¹ h⁻¹^(c)g ethanol g xylose consumed⁻¹^(d)g xylitol g xylose consumed⁻¹^(e)g ethanol g cells⁻¹ h⁻¹

TABLE 3 Specific XI activities in cell extracts, measured at 50° C.Strain 50° C. TMB 3044 + XI (YEplacHXT-XI) 0.188 ± 0.017 TMB 3044 +plasmid YEplac195 0.004 ± 0.003 TMB 3050 (grown in glucose) 0.095 ±0.017 TMB 3050 (grown in xylose) 0.153 ± 0.031

FIGURE LEGENDS

FIG. 1. Aerobic growth of mutant strain TMB 3050( ) and parental strainTMB 3044 with XI (Δ) in defined mineral medium with 50 g/l xylose as thesole carbon source. TMB 3044 with XI was pre-cultured in defined mineralmedium containing glucose and TMB 3050 was pre-cultured in definedmineral medium containing xylose.

FIG. 2. The gene construct of the present strain

1. A saccharomyces cerevisiae strain utilizing xylose for fermentingethanol expressing xylose isomerase (XI), overexpressing xylulokinase(XK), overexpressing the pentose phosphate pathway (PPP), andnon-expressing aldose reductase (AR) and being adapted to growth inmineral defined medium with xylose as sole carbon source.
 2. Asaccharomyces cerevisiae strain according to claim 1, wherein the strainexpresses xylose isomerase derived from a Thermus thermophilus xylAgene, overexpresses xylulokinase by an addition of a plasmid YIpXK (Lönnet al. 2003) linearized with NdeI coding for xylulokinase, overexpressesthe pentose phosphate pathway by adding the genes TAL1, TKL1, RPE1,RKI1, and non-expresses aldose reductase by deletion of the gene GRE3and being adapted to growth in mineral defined medium with xylose assole carbon source.
 3. A saccharomyces cerevisiae strain according toclaim 1, wherein the strain exhibits a growth rate of at least 0.12 h⁻¹,and a xylose uptake rate of at least 0.10 g xylose/g cells/h.
 4. ASaccharomyces cerevisiae strain according to claim 1, wherein the strainis TMB3050 deposited at Deutsche Sammlung von Mikroorganismen undZellkulturen under deposition number DSM 15834.